Recommendations for the clinical interpretation and reporting of copy number gains using gene panel NGS analysis in routine diagnostics

Astrid Eijkelenboom,Maartje J Vogel,Carel J M Van Noesel,Arja Ter Elst,Manon M H Huibers,Léon C Van Kempen ,Wendy W.j De Leng ,A J C Van Den Brule ,Winand N.m Dinjens ,Ed Schuuring,Cornelis J J Huijsmans,Anke Van Den Berg,Bastiaan B.j Tops ,Esther Korpershoek,Hendrikus J Dubbink,Ernst‐Jan M Speel ,Daniëlle A.m Heideman ,Tom Van Wezel,Willemina R.r Geurts-Giele ,Judith Jeuken ,Patricia J.t.a Groenen ,Floris H Groenendijk,Leonie I Kroeze,Petra M Nederlof,Marjolijn J L Ligtenberg

doi:10.1007/s00428-019-02555-3

Abstract

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity.

Highlights

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to identify actionable copy number gains in addition to sequence variants
Diagnostic NGS gene panels allow parallel detection of high-level gene amplifications associated with targeted therapy
Note that at this moment Bbest practices^ are yet to be determined and the choice of method and software is generally based on in house availability and validation. These approaches start with sample normalization to correct for differences in total reads, which is especially required in the context of formalin-fixed paraffin-embedded (FFPE) tissue analyses with variable input quality and quantity of genomic DNA (gDNA)

Summary

Introduction

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to identify actionable copy number gains (gene amplifications) in addition to sequence variants. Awareness of assay limitations is critical for routine diagnostics (i) Affected by thresholds and neoplastic cell percentage (ii) Should be included in clinical report tyrosine kinase inhibitor (TKI) crizotinib a FISH established cutoff of 10 copies of the MET gene in lung adenocarcinoma has been suggested [16], while only very limited data on the response related to low-level (4–9 copies) and high-level (≥ 10 copies) amplification are presently available [17, 18].

Results

Conclusion